Our long-term objective is to understand the molecular events that underlie the induction of differentiation in ML-1 human myeloblastic leukemia cells. We have identified a gene that increases in expression early in induction with 12-0-tetradecanoylphorbol-13-acetate (TPA). We have designated this gene mcl-1, based on its isolation from maturing myeloid ML-1 cell leukemia. The increase in mcl-1 mRNA occurs within one to three hours, preceding maturation to monocyte/macrophages at one to three days. We found mcl-1 to exhibit significant homology to bcl-2, a gene that plays an as-yet poorly understood role in t(14;18)B-cell lymphomas. mcl-1 is the first example of a cellular gene with significant homology to bcl-2. We now propose to study the mcl-1-encoded protein, exploring its possible site(s) of action and role(s) in cell proliferation, differentiation, and viability. We will study the behavior of the mcl-1 protein in cell-free systems (Aim I), determining whether it is processed by microsomal membranes (via its putative signal sequence) or might associate with isolated mitochondria (via the potential membrane spanning region with homology to bcl-2). We will prepare monoclonal and polyclonal antibodies (Aim II) for use in studying the mcl-1 protein in intact cells (Aim III): Using results from Aim I as a guide, we will assess the intracellular localization of mcl-1 (Aim IIIA). We will also determine how expression of the mcl-1 protein relates to hematopoietic cell lineage, maturation stage, and proliferation status (Aim IIIB). Finally, using electroporation, we will test for effects of mcl-1 on cell viability or mitochondrial function (Aim IIIC). It will be interesting to compare mcl-1 to bcl-2, which localizes to mitochondria, is expressed in immature cells of the lymphoid lineage, and can block apoptosis. mcl-1, bcl-2, and a related viral gene BHRF-1, may define a family of genes different from other known families including oncogenes. The studies proposed here should yield information basic to elucidating the function of mcl-1; they may also advance us towards an integrated understanding of this emerging gene family.